Sulforaphane ameliorates bisphenol A-induced hepatic lipid accumulation by inhibiting endoplasmic reticulum stress

The aim of the present study was to investigate the role of endoplasmic reticulum (ER) stress in bisphenol A (BPA) – induced hepatic lipid accumulation as well as the protective effects of Sulforaphane (SFN) in this process. Human hepatocyte cell line (LO2) and C57/BL6J mice were used to examine BPA-triggered hepatic lipid accumulation and the underlying mechanism. Hepatic lipid accumulation, triglycerides (TGs) levels, the expression levels of lipogenesis-related genes and proteins in the ER stress pathway were measured. It was revealed that BPA treatment increased the number of lipid droplets, the levels of TG and mRNAs expression of lipogenesis-related genes, and activated the ER stress pathway. These changes were inhibited by an ER stress inhibitor 4-phenylbutyric acid. SFN treatment abrogated BPA-altered hepatic lipid metabolism and ameliorated BPA-induced ER stress-related markers. Together, these findings suggested that BPA activated ER stress to promote hepatic lipid accumulation, and that SFN reversed those BPA effects by alleviating ER stress.

www.nature.com/scientificreports/ each group was measured every week. The mice were euthanized after the last BPA dose treatment , and the liver tissues were collected for further examination.
Histological analysis. The liver tissues were embedded in paraffin after treatment with a 10% formaldehyde solution overnight. H&E staining of the tissue slides was performed for the examination of the tissue morphology. The Panoramic Scanner (3DHISTECH, Budapest, Hungary) was used to analyze the liver slices.

RNA isolation and quantitative real-time PCR (qRT-PCR).
TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract the total RNA from the cells and liver tissues. The mRNA levels of peroxisome proliferator-activated receptor gamma (PPARγ), sterol regulatory element-binding transcription factor 1 (SREBF1), fatty Acid Synthase (FASN) and diacylglycerol-O-acyltransferase-1 (DGAT-1) were determined by qRT-PCR. Polymerase chain reaction primers used in this study are listed in Table 1.
Western blotting. After the appropriate treatments, the liver tissues and cells were collected and lysed in RIPA buffer containing 1X protease inhibitor cocktail (Pierce) and EDTA. The protein content of the total protein lysates was determined by the Bradford Protein Assay Kit. On a 10% SDS-PAGE, equal amounts of 60 μg of proteins were separated and transferred to PVDF membranes (Bio-Rad). After blocking with 5% nonfat dry milk, the membranes were incubated with suitable primary antibodies (1:500-1:1000 dilution) at 4 °C on a rotating shaker overnight. After flushing in Tris-buffered saline/Tween (TBS/T), the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:10,000 dilution) at room temperature for 1 h. GAPDH was used as a loading control to quantitatively compare the expression of proteins. ImageJ software (National Institutes of Health, Bethesda, MD) was used to quantify the protein bands on blots for densitometric analyses.
Statistical analysis. The mean and standard deviation of all experimental data were determined. Unpaired t-test and one-way ANOVA analyses were employed to examine the statistical differences between two or multiple groups. GraphPad Prism 7.0 software (USA, San Diego, CA) was used for statistical analysis and bar graph generation. p < 0.05 was considered to indicate statistical significance.  36 . A study reported that serum BPA concentrations in women with miscarriages were as high as 47.7 ng/mL (209.9 nM) 37 . In our previous study which investigated BPA-induced abnormalities in insulin signaling pathway and glucose metabolism in hepatocytes, we observed that the effect of BPA at 100 nM was the most significant among the three exposure concentrations of 1, 10, and 100 nM 38 . Therefore, based on above information, 100 and 1000 nM BPA concentrations were used in the subsequent experiments. As shown in Fig. 1b,c, BPA exposure promoted hepatic lipid accumulation, resulting in dramatic LDs (marked green with BODIPY493/503 staining) and higher TG levels ( Fig. 1d). BPA treatment did not change TC levels considerably (Fig. 1e) (p > 0.05). We then evaluated the mRNA expression of the following lipogenesis-related genes in LO2 cells: PPARγ, SREBF1, FASN, and DGAT-1. It was shown that the mRNA expression levels of those genes were upregulated in BPA groups ( Fig. 1f-i). These results suggest that BPA can induce lipid accumulation in human-immortalized liver LO2 cells.

BPA induces hepatic lipid accumulation by activating ER stress in LO2 cells. After treatment
with BPA for 24 h, the levels of ER stress pathway proteins, including p-eIF2α, CHOP, and XBP1s, were determined. As expected, BPA treatment considerably increased p-eIF2α, CHOP, and XBP1s levels ( Fig. 2a,b). These data suggested that BPA impeded ER homeostasis. 4-PBA is an ER stress inhibitor with low-molecular-weight  www.nature.com/scientificreports/ that can stabilize the conformation of proteins, improve the folding capacity of the ER, and facilitate the trafficking of mutant proteins to suppress ER stress 39 . In this study, 4-PBA was used to inhibit ER stress signaling and to examine the role of ER stress in BPA-induced lipid accumulation in LO2 cells. The western blotting results showed that the levels of p-eIF2α, CHOP, and XBP1s were reduced in cells pre-treated with 4-PBA, suggesting that 4-PBA inhibited ER stress (Fig. 2c,d). Furthermore, 4-PBA prevented BPA-induced upregulation of p-eIF2α, CHOP, and XBP1s (Fig. 2c,d). Meanwhile, BPA-triggered hepatic lipid accumulation was diminished in 4-PBA-pretreated cells (Fig. 2e,f). Similar results were also observed for TG levels (Fig. 2g) and the mRNA levels of the lipogenesis-related genes ( Fig. 2h-k). Collectively, these data suggest that ER stress plays a regulatory role in lipid metabolism disorder induced by BPA.  (Fig. 3b-d). Simultaneously, SFN down-regulated the mRNA expression of BPA-induced lipogenesis-related genes and the levels of ER stress markers (Fig. 3e-j). These results suggest that SFN improves abnormal lipid metabolism and ER stress caused by BPA in LO2 cells. www.nature.com/scientificreports/ SFN attenuates BPA-induced hepatic lipid accumulation and ER stress in mice. We then verified our in vitro experiments' results in vivo by randomly dividing C57/BL6J mice into three groups according to body weight as follows: control, BPA, and SFN-intervention groups. After treatment, the body weights and liver weights of mice were measured. No considerable differences were observed in body weights among the three groups (Fig. 4a). BPA considerably increased the liver/body weight ratio (Fig. 4b) (p < 0.01). We further investigated whether BPA treatment promoted hepatic lipogenesis in these mice. H&E staining showed abnormal LDs in BPA-treated mouse liver tissues, which was attenuated by SFN treatment (Fig. 4c). Furthermore, we detected the expression of lipogenesis-related genes in mouse livers. As shown in Fig. 4d-f, BPA upregulated lipogenesisrelated gene expression, whereas SFN downregulated their expression. In terms of the ER stress pathway, it was revealed that BPA treatment elevated ER stress marker's levels in mouse livers (Fig. 4g,h), and SFN suppressed the activation of ER stress pathway. These results suggest that BPA exposure promotes ER stress and lipid accumulation in the mouse liver, and these effects were diminished by SFN.

Discussion
The present study illustrated that BPA promoted lipid accumulation and upregulated the expression of lipogenesis-related genes in human hepatocyte LO2 cells and mouse livers by activating the ER stress pathway, which was ameliorated by blocking ER stress or SFN treatment. The liver is the primary target organ of BPA. It is also the most important organ for lipid synthesis and plays a key role in maintaining lipid metabolic homeostasis. Previous studies in animals and humans have shown that exposure to BPA may increase the accumulation of lipids in hepatocytes and the levels of key enzymes in lipid synthesis 40,41 . However, the molecular mechanisms by which BPA causes metabolic abnormalities in hepatic lipids remain to be elucidated. In the present study, we also found that BPA increased hepatic accumulation of lipids, TG levels, and the mRNA levels of lipogenesis-related genes. However, we did not observe differences in cholesterol levels in LO2 cells treated with BPA. In contrast, a study reported by Li et al. showed that hepatic cholesterol content was increased when C57BL/6 mice and HepG2 cells exposed to BPA 42,43 . This suggests that the effects of BPA on specific lipids may differ, which might be related to factors such as patients, BPA doses, and treatment duration, etc.
SREBF1 is a membrane-bound transcription factor that activates the expression of most genes required for hepatic lipogeneses, such as ACC, FAS, and SCD1 44,45 . PPARγ is an additional reinforcing lipogenic signal that assists sterol regulatory element-binding protein-1c in triggering hepatic steatosis development 46 . PPARγ is The levels of (i) p-eIF2α, CHOP, and (j) XBP1s were measured by Western blotting. The blots of p-eIF2α and GAPDH were cropped from different portions of the same membrane. CHOP blot was not cropped from the same membrane as the above two blots but from the same sample. The blots of XBP1s and GAPDH were cropped from different parts of the same membrane. The original blots are presented in Supplementary Figs. S5, 6. Data are expressed as the mean ± SD of 3 independent experiments. *p < 0.05 and **p < 0.01, when compared with the control group; #p < 0.05 and ##p < 0.01, when compared with the BPA group. www.nature.com/scientificreports/ also involved in regulating DGAT-1, which catalyzes the final step in triacylglycerol synthesis 47 . We speculated that BPA might regulate lipid metabolism by affecting the SREBF1 and PPARγ pathway. In the present study we found that the mRNA levels of SREBF1, PPARγ, FASN and DGAT-1 in LO2 cells and mice liver tissues treated with BPA were markedly increased. These results suggest the mechanism by which BPA increased lipid synthesis may be related to the activation of SREBF1 and PPARγ pathway. The expression of (g) p-eIF2α, CHOP, and (h) XBP1s was measured by Western blotting. The blots of p-eIF2α, CHOP, and GAPDH were not cropped from the same membrane, but rather from the same sample. The blots of XBP1s and GAPDH were not cropped from the same membrane, but rather from the same sample. The original blots are presented in Supplementary Figs www.nature.com/scientificreports/ A literature search revealed that the mechanistic studies of BPA-induced liver damage mainly focused on mitochondrial dysfunction, ROS formation, and inflammatory states [48][49][50] , while relative less studies were available regarding ER stress. The ER, an intracellular membranous network structure that integrates cellular signaling and homeostasis, is also the primary location for lipid production in hepatocytes 14 . Some conditions, such as glucose and energy deprivation, high cholesterol, and protein glycosylation inhibition, can activate ER stress [51][52][53] . ER stress can cause de novo lipogenesis, directly affecting hepatic lipid metabolism 14 . Upon ER stress, ER membranes is cleaved and the released SREBF enter the nucleus, activating the promoter of sterol biosynthesis, thus increasing fatty acid, triglycerides and cholesterol synthesis 54,55 . Therefore, we hypothesized that BPA induced hepatic lipogenesis by activating the ER stress pathway to enhance SREBF signaling. In LO2 cells treated with BPA, we found that BPA activated the ER stress pathway by increasing p-eIF2α, CHOP, and XBP1s levels; however, BPAinduced lipid metabolism abnormality was reversed by ER stress inhibitor 4-PBA, suggesting the important role of ER stress in BPA-triggered hepatic lipid abnormality. In the animal experiment, the levels of ER stress markers in the liver of BPA-treated mice were also increased considerably. Previous studies showed that BPA treatment induced lipid accumulation in the liver and enhanced ER stress in ovariectomized mice fed on a high-fat diet, and in Watanabe heritable hyperlipidemic rabbits 18,56 . Nevertheless, the present results illustrated for the first time that BPA treatment alone affected liver fat metabolism and ER stress in vivo and in vitro. These results suggest that low-dose BPA may interfere with hepatic lipid metabolism and active ER stress, even if it does not interact with factors such as a high-fat diet.
Recent studies have shown that SFN can improve lipid accumulation and the levels of key lipogenic enzymes in hepatocytes and liver tissues induced by high-fat diet, through the mechanisms mainly involving oxidative stress, inflammation, and insulin resistance [57][58][59] . One study showed that SFN inhibited the expression of ACC1 and SCD1 by targeting the ERS response pathway, thereby improving high-fat-diet-induced abnormal hepatic lipid metabolism 60 . Modulation of the ER stress response pathway via the nuclear factor erythroid 2-related factor 2-regulated antioxidant pathway contributed to the action of SFN 22 . In the present study we hypothesized that SFN improved BPA-induced lipid metabolism by inhibiting the ER stress pathway. We depicted that SFN ameliorated the accumulation of lipids in hepatocytes and the levels of key lipogenic enzymes through inhibition of the ERS pathway. In in vivo experiment, however, SFN treatment did not resulted in considerable decrease in the liver/body weight ratio compared with BPA group, which might be related to the relative short intervention duration of SFN. Nevertheless, the present study is the first investigation to reveal the ameliorative effect of SFN on BPA-induced lipid metabolic abnormalities and ER stress in both in vitro and in vivo settings. In conclusion, our findings suggested that BPA activated ER stress to promote hepatic lipid accumulation, and that SFN diminished BPA-triggered effects on lipid metabolic abnormalities by alleviating ER stress.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.